Morphology and differentiation of radial glia in the developing rat spinal cord

Important events underlying the proper functioning of the central nervous system (CNS) include the production, assembly, and differentiation of appropriate types and numbers of cells during development. The mechanisms that control these events are difficult to unravel because of displacement of cells from their sites of origin to their permanent locations and because of the diverse cellular composition of the CNS. As in other regions of the mammalian CNS, the two major classes of neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. In the developing spinal cord, radial glia are prominent. In this study, radial glia in the cervical region of the spinal cord were analysed. 1,1'Dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) was used to determine the morphology and distribution of radial glia during spinal cord development. The DiI labelling technique enabled locating glial precursor cells during spinal cord development. Radial fibres that extended from the central canal to the pial surface were present at embryonic days 14, 16, and 18 in the developing spinal cord. Their distribution was restricted with increasing development, and by embryonic day 20 the only remaining evidence of radial glia were short radial processes in the white matter.     

Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin-positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self-renewing radial cells, distinct from an earlier pseudo-stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP-positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.     

Distribution of radial glia in the developing telencephalon of chicks

Radial glia are known to have a sparse and uneven distribution in the telencephalon of adult birds. The present study utilizes antibodies against vimentin to reveal a more extensive, and more clearly radial, set of radial glia in the chicken telencephalon during the first half of embryogenesis. This initially extensive radial glial fiber system becomes distorted and reduced between 10 and 14 days of incubation. This reduction coincides with the cytoarchitectural differentiation of the telencephalon into its major adult subdivisions. Because developing neurons tend to migrate along radial glial fibers in both birds and mammals, a topological projection of these major subdivisions onto the embryonic ventricular zone along the radial glial fibers suggests hypotheses about lineage relationships that can be tested by subsequent experimental methods. This analysis suggests that the major components of the avian dorsal ventricular ridge, i.e., the ventral hyperstriatum, the neostriatum with its various subdivisions, part of the archistriatum, and probably also the piriform cortex, all derive from overlapping portions of the lateral pallial ventricular zone. Staining with antibodies against neurofilament suggests that this developmental parcellation of the lateral pallial complex is associated with the development of neuronal fiber systems. J. Comp. Neurol. 387:399–420, 1997. © 1997 Wiley-Liss, Inc.     

Developmental potential of radial glia investigated by transplantation into the developing rat ventricular system in utero

During development there is a clear correlation between position of dividing progenitor cells, mode of division and developmental potential, suggesting that the local environment of progenitor cells may influence their cell fate [ 17 (6), 639-647]. The contribution of these conditions was investigated here by transplantation of radial glial progenitor cells into isotopic, isochronic, heterotopic and heterochronic environment conditions. Neuronal cells were removed from E14 spinal cords using negative immunoselection. The remaining radial glia were transplanted into the ventricular system of host embryos and pups. Distance of migration as well as morphological and antigenic phenotype of transplanted radial glia was examined after various survival times post transplantation. Host age clearly influenced migration and differentiation of transplant cells, with transplant cells migrating further in younger hosts and differentiating earlier in older aged host environments. Evidence is presented showing that most transplanted spinal cord radial glia give rise to astrocytes. In addition some transplanted radial glia were shown to give rise to neurons in spinal cord regions. Radial glia did not appear to generate neurons in the brains of host animals until postnatal ages, perhaps because transplanted radial glia were isolated from spinal cord and thus may not have been influenced to behave as endogenous radial glia in the brain which commonly produce neurons.     

Radial glia and somal translocation of radial neurons in the developing cerebral cortex

A series of recent studies have demonstrated that radial glia are neural precursors in the developing cerebral cortex. These studies have further implied that these cells are the sole precursor constituents of the dorsal forebrain ventricular zone that generate the projection neurons of the cortex. In view of these new findings, this review discusses radial neurons, a progeny of cortical neurons that are generated by radial glia and adopt somal translocation as the mode of migration.     

Guidance of callosal axons by radial glia in the developing cerebral cortex

During development, columns of the mammalian cerebral cortex are formed by migration of neurons along fascicles of radial glia. Subsequently, axons of the corpus callosum connect reciprocal regions of each cerebral hemisphere. To determine whether the radial growth of callosal afferents through the developing cortex may be guided by particular cellular elements, we examined the ultrastructural relationship between callosal afferents and radial fibers in the early postnatal hamster sensorimotor cortex. Developing callosal axons and their growth cones were labeled with HRP injected into the cortex at 3 d postnatal when the growth cones have extended across the callosum and are just entering the contralateral cortex. An EM analysis of 30 HRP-labeled axons and their growth cones revealed that they extended upon fascicles of radial processes associated with migrating neurons. Reconstruction of seven of these growth cones, serially sectioned in their entirety, showed that growth cones were associated with the same radial fascicle as their axon. Growth cones also touched other cellular elements such as axons. However, the finding that callosal afferents, from the point at which they enter the cortex to their growth cones, were apposed to a continuous fascicle of radial fibers suggests that callosal axons are tracking along radial processes. We conclude that the majority of the radial processes within fascicles are likely to be glial, based on their relatively large diameters, electron-lucent cytoplasm with a regular array of microtubules, the presence of glycogen granules, occasional cytoplasmic protrusions lacking microtubules, and their consistent association with migrating neurons. We propose therefore that radial glia may serve a guidance function for growing callosal axons in their radial trajectory through the developing cerebral cortex.     

Afadin controls cell polarization and mitotic spindle orientation in developing cortical radial glia

Background

In developing tissues, cell polarity and tissue architecture play essential roles in the regulation of proliferation and differentiation. During cerebral cortical development, adherens junctions link highly polarized radial glial cells in a neurogenic niche that controls their behavior. How adherens junctions regulate radial glial cell polarity and/or differentiation in mammalian cortical development is poorly understood.

Results

Conditional deletion of Afadin, a protein required for formation and maintenance of epithelial tissues, leads to abnormalities in radial glial cell polarity and subsequent loss of adherens junctions. We observed increased numbers of obliquely-oriented progenitor cell divisions, increased exit from the ventricular zone neuroepithelium, and increased production of intermediate progenitors.

Conclusions

Together, these findings indicate that Afadin plays an essential role in regulating apical-basal polarity and adherens junction integrity of radial glial cells, and suggest that epithelial architecture plays an important role in radial glial identity by regulating mitotic orientation and preventing premature exit from the neurogenic niche.

     

BMP and LIF signaling coordinately regulate lineage restriction of radial glia in the developing forebrain

The earliest radial glia are neural stem cells that guide neural cell migration away from ventricular zones. Subsequently, radial glia become lineage restricted during development before they differentiate into more mature cell types in the CNS. We have previously shown that subpopulations of radial glial cells express markers for glial and neuronal restricted precursors (GRPs and NRPs) in expression patterns that are temporally and spatially regulated during CNS development. To characterize further the mechanism of this regulation in rat forebrain, we tested whether secreted factors that are present during development effect lineage restriction of radial glia. We show here that in radial glial cultures LIF/CNTF up-regulates, whereas BMP2 down-regulates GRP antigens recognized by monoclonal antibodies A2B5/4D4. These activities combined with secretion of BMPs dorsally and LIF/CNTF from the choroid plexus provide an explanation for the graded distribution pattern of A2B5/4D4 in dorso-lateral ventricular regions in vivo. The regulation by LIF/CNTF of A2B5/4D4 is mediated through the JAK-STAT pathway. BMP2 promotes expression on radial glial cells of the NRP marker polysialic acid most likely by regulating N-CAM expression itself, as well as at least one polysialyl transferase responsible for synthesis of polysialic acid on N-CAM. Taken together, these results suggest that generation of lineage-restricted precursors is coordinately regulated by gradients of the secreted factors BMPs and LIF/CNTF during development of dorsal forebrain.     

Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebrum

Microchemical techniques and histologic examination were used to study the somatosensory area of developing cerebral cortex and subcortical white matter in rats made thyroid hormone deficient immediately after birth. The body weight of these animals failed to show the normal growth spurt, achieving levels which were only 40% of age-matched controls by 50 post-natal days. The endocrine imbalance initiated by a lack of thyroid hormone from the day of birth results in a metabolic derangement which disturbs the migration of glial cells from the subependymal zone into cortex between 10 and 50 days of post-natal life. During this same developmental period, the differentiation of neurons is impaired, resulting in decreased formation of axodendritic processes and synapses. Finally, in association with the pathologic development of axons and fewer numbers of well-differentiated interfascicular oligodendroglia, there is a failure of myelin formation. Hence, in rats made hypothyroid at birth, the cerebrum is subjected to drastic alterations in the normally continuous and interdependent process of neuron and glial cell differentiation.     

Signaling to and from radial glia

Radial glia represent the major glial cell type in the developing CNS and perform many essential functions, which range from acting as neural precursors to providing physical substrates for newborn neurons to migrate on. Previous work has shown that cell-cell signaling is important for the development of the radial glial phenotype. In particular, signals from newborn neurons appear to contribute significantly to the formation of this cell type. In addition, radial glia may be involved in reciprocal signaling roles that contribute to regional patterning and differentiation in the developing CNS.     

Substrates and routes of migration of early generated neurons in the developing rat thalamus

We investigated the substrates supporting neuronal migration, and its routes, during early thalamic development in the rat. Neurons and axonal and glial fibres were identified in embryos with single and double immunohistochemistry; dynamic data were obtained with cell tracers in short-term organotypic cultured slices. The earliest thalamic neurons, originating from the ventricular neuroepithelium between embryonic days 13 and 15, include those of the reticular thalamic nucleus. At this developmental stage, calretinin, calbindin or gamma-aminobutyric acid immunostaining revealed both radially and nonradially orientated neurons in the region of reticular thalamic migration, between the dorsal and ventral thalamic primordia. In cultured slices, injections of fluorescent dyes in the neuroepithelium labelled neurons in a migratory stream along radial glia in the same zone. Some labelled fusiform cells departed from this radial trajectory along orthogonal routes within the dorsal thalamus. Confocal microscopy revealed nonradially orientated neurons in close apposition with a fibre system parallel to the lateral thalamic surface. These fibres expressed axonal markers, including the intermediate filament protein alpha-internexin and a polysialylated form of neuronal cell adhesion molecule. Active migration of nonradially orientated neurons along neuronal substrates was confirmed in living cultured slices. In addition, in vitro and ex vivo experiments revealed neurons migrating tangentially in association with glial fibres. These results provide novel evidence that: (i) early generated thalamic neurons follow nonradial routes in addition to glia-linked radial migration; and (ii), nonradially migrating thalamic neurons move along both glial and axonal substrates, which could represent a distinctive feature of thalamic development.     

PSA-NCAM in the developing and mature thalamus

The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is involved in several morphogenetic processes of the central nervous system. In the present study the expression of PSA-NCAM has been investigated in the rat thalamus during embryonic and postnatal development using light and electron microscopic immunocytochemical techniques. At all the examined ages, PSA-NCAM staining in the thalamus was mainly observed along neuronal plasmatic membranes and absent in astrocytes identified by labelling with cytoskeletal (vimentin and glial fibrillary acidic protein) and membrane (GABA transporter-3) markers. At embryonic day 14 the immunoreactivity was restricted to the dorsal thalamic mantle and to the region of reticular thalamic migration and subsequently it extended throughout the whole thalamic primordium. PSA-NCAM labelling remained intense and homogeneously distributed along perinatal period, but from P4 it began to decrease selectively, persisting throughout adulthood only in the reticular nucleus, ventral lateral geniculate nucleus and midline and intralaminar nuclei. The expression of this adhesion molecule differed in areas characterized by the presence of neurons containing distinct calcium binding proteins, as PSA-NCAM labelling was intense around calretinin-positive neurons, whereas it decreased in some calbindin-immunoreactive regions. These findings show evidence of a selective neuronal expression of PSA-NCAM in developing thalamus, supporting its suggested role in cell migration and synaptogenesis as it occurs in the cerebral cortex. In adulthood PSA-NCAM could instead be a marker of thalamic nuclei that retain a potential for synaptic plasticity.     

Immunohistochemical observations on rat radial glia: relationship with the origin of ethylnitrosourea-induced tumors

Summary Gliomas induced in the rat by transplacental administration of ethylnitrosourea (ENU) are intensely immunoreactive for vimentin and scarcely for glial fibrillary acidic protein (GFAP). Since tumoral transformation takes place during the late fetal and early postnatal period, the sequential expression of the two glial antigens has been investigated in this age period in ENU-treated and control rats. Immunohistochemical and immunoelectron microscopical methods have been employed. Vimentin was widely expressed starting from embryonal day 14 (E 14) in the processes of radial glia; as long as radial glia was present, vimentin decorated it. GFAP was, at earliest, observed at E 20 and expressed by glial cells with a stellate, i.e., mature shape. No GFAP-positive radial process was observed. No difference was found between ENU-treated and control rats. Since ENU is most effective in producing tumors when administered at the 16–17th day of fetal life, vimentin-positive radial glia is a candidate target of ENU. The similarity of intermediate filament pattern between radial glia in the late fetal life and tumors induced by transplacental ENU suggests that radial glia might be the cell of origin.Supported in part by M. P. I. 60% grant, Rome     

The history of radial glia.

Radial glial cells are now recognized as a transient population that serves as scaffolding for neuronal migration. The recognition of the existence and role of radial glia has not been smooth, and here we provide a brief historical overview on the pioneering studies on this subject. The histologists and embryologists Albert K?lliker and Wilhelm His performed seminal investigations on cortical morphogenesis in the last decades of the 19th century. However, the introduction of the silver impregnation Golgi technique, and its diffusion in the late 1880s, played a crucial role in the detection of radial glial processes. The radial arrangement of fibers emerging from the neuroepithelium lining the central canal was initially detected in the embryonic spinal cord by Camillo Golgi himself. The first Golgi impregnation of the cerebral cortex of mammalian fetuses was performed by Giuseppe Magini, who detected radial fibers extending from the ventricular neuroepithelium, and observed cells intercalated along these processes. Radial fibers, regarded as epithelial or ependymal processes, were then observed in the developing spinal cord and cerebral cortex by several investigators. Santiago Ramón y Cajal was the first to suggest that radial fibers were modified astrocytic processes functioning as a support during cortical histogenesis. Cajal acknowledged Magini's findings, but he criticized Magini's observations on the existence of neurons along radial fibers. With the advent of electron microscopy, the existence of radially arranged glial processes along which young neurons migrate was finally ascertained in the early 1970s by Pasko Rakic, thus opening a new era in the cellular and molecular biology of radial glia.     

Adenylyl cyclases: expression in the developing rat thalamus and their role in absence epilepsy

Adenylyl cyclases (ACs) synthesize the second messenger cyclic AMP (cAMP) which influences the function of multiple ion channels. Former studies point to a malfunction of cAMP-dependent ion channel regulation in thalamocortical relay neurons that contribute to the development of the absence epileptic phenotype of a rat genetic model (WAG/Rij). Here, we provide detailed information about the thalamic gene and protein expression of Ca²⁺/calmodulin-activated AC isoforms in rat thalamus. Data from WAG/Rij were compared to those from non-epileptic controls (August-Copenhagen Irish rats) to elucidate whether differential expression of ACs contributes to the dysregulation of thalamocortical activity. At one postnatal stage (P21), we found the gene expression of two specific Ca²⁺-activated AC isoforms (AC-1 and AC-3) to be significantly down-regulated in epileptic tissue, and we identified the isoform AC-1 to be the most prominent one in both strains. However, Western blot data and analysis of enzymatic AC activity revealed no differences between the two strains. While basal AC activity was low, cAMP production was boosted by application of a forskolin derivative up to sevenfold. Despite previous hints pointing to a major contribution of ACs, the presented data show that there is no apparent causality between AC activity and the occurrence of the epileptic phenotype.     

Organization and metamorphosis of glia in the Drosophila visual system

The visual system of Drosophila is an excellent model for determining the interactions that direct the differentiation of the nervous system's many unique cell types. Glia are essential not only in the development of the nervous system, but also in the function of those neurons with which they become associated in the adult. Given their role in visual system development and adult function we need to both accurately and reliably identify the different subtypes of glia, and to relate the glial subtypes in the larval brain to those previously described for the adult. We viewed driver expression in subsets of larval eye disc glia through the earliest stages of pupal development to reveal the counterparts of these cells in the adult. Two populations of glia exist in the lamina, the first neuropil of the adult optic lobe: those that arise from precursors in the eye-disc/optic stalk and those that arise from precursors in the brain. In both cases, a single larval source gives rise to at least three different types of adult glia. Furthermore, analysis of glial cell types in the second neuropil, the medulla, has identified at least four types of astrocyte-like (reticular) glia. Our clarification of the lamina's adult glia and identification of their larval origins, particularly the respective eye disc and larval brain contributions, begin to define developmental interactions which establish the different subtypes of glia.     

Nitric oxide synthase in the adult and developing thalamus: Histochemical and immunohistochemical study in the rat

The distribution of neuronal elements that express nitric oxide synthase (NOS), the synthetic enzyme of the free radical nitric oxide, was investigated in the adult and developing rat thalamus by means of NADPH-diaphorase (NADPH-d) histochemistry, which is a marker of NOS. Immunocytochemistry was also used to confirm the equivalence between the histochemical pattern of staining and the distribution of the expression of the neuronal NOS isoform. In the adult thalamus, NADPH-d-positive and NOS-immunoreactive perikarya were selectively concentrated along the midline (in the paraventricular, rhomboid, and central medial nuclei) and in the dorsal and ventral lateral geniculate nuclei. Isolated clusters of stained neurons were also observed in the lateral posterior nucleus, in the dorsal part of the medial geniculate nucleus, and in the ventromedial nucleus. Positive perikarya were either absent or very sparse in the other thalamic nuclei. Many thalamic domains were, however, characterized by distinct patterns of NADPH-d-positive fibers, preterminal and terminal-like elements. The highest density of stained neuropil was observed in the anteroventral and anteromedial nuclei, in several of the midline nuclei, in the anterior intralaminar nuclei, and in the lateral and medial geniculate nuclei. Although histochemical reactivity was observed in the thalamus at birth, the intensity and the pattern of distribution of staining observed in adulthood was not achieved until the end of the third postnatal week. The NADPH-d histochemical positivity followed discrete developmental schedules in various thalamic domains, and different areas reached a mature pattern at different ages. In addition, populations of transiently stained neuronal cell bodies were observed in the medial thalamus during the first two postnatal weeks. These results show discrete patterns of expression of NOS in the adult and developing thalamus and suggest that nitric oxide may be involved in selected physiological and developmental roles in different thalamic domains. J. Comp. Neurol. 388:89–105, 1997. © 1997 Wiley-Liss, Inc.     

Specific characteristic of radial glia in the human fetal telencephalon

Phenotypic characteristics of cells in the developing human telencephalic wall were analyzed using electron microscopy and immunocytochemistry with various glial and neuronal cell markers. The results suggest that multiple defined cell types emerge in the neocortical proliferative zones and are differentially regulated during embryonic development. At 5-6 weeks gestation, three major cell types are observed. Most proliferating ventricular zone (VZ) cells are labeled with radial glial (RG) markers such as vimentin, glial fibrillary acidic protein (GFAP), and glutamate astrocyte-specific transporter (GLAST) antibodies. A subpopulation of these RG cells also express the neuronal markers beta III-tubulin, MAP-2, and phosphorylated neurofilament SMI-31, in addition to the stem cell marker nestin, indicating their multipotential capacity. In addition, the presence of VZ cells that immunoreact only with neuronal markers indicates the emergence of restricted neuronal progenitors. The number of multipotential progenitors in the VZ gradually decreases, whereas the number of more restricted progenitors increases systematically during the 3-month course of human corticogenesis. These results suggest that multipotential progenitors coexist with restricted neuronal progenitors and RG cells during initial corticogenesis in the human telencephalon. Since the multipotential VZ cells disappear during the major wave of neocortical neurogenesis, the RG and restricted neuronal progenitors appear to serve as the main sources of cortical neurons. Thus, the diversification of cells in human VZ and overlying subventricular zone (SVZ) begins earlier and is more pronounced than in rodents.